GSK3β-driven SOX2 overexpression is a targetable vulnerability in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest forms of human malignancy that currently lacks approved targeted therapeutics. Accumulating evidence suggests that SOX2 overexpression is a key driving factor for ESCC and various squamous cell carcinoma. Here, through screening a small-molecule kinase inhibitor library, we identified GSK3 ss as a kinase that is critically required for robust SOX2 expression in ESCC cells. GSK3 ss did not promote SOX2 transcriptionally but was required for SOX2 protein stability. We demonstrated that GSK3 ss interacts with and phosphorylates SOX2 at residue S251, which blocks SOX2 from ubiquitination and proteasome-dependent degradation instigated by ubiquitin E3 ligase CUL4A(DET1-COP1). Pharmacological inhibition or knockdown of GSK3 ss by RNA interference selectively impaired SOX2-positive ESCC cell proliferation, cancer stemness, and tumor growth in mouse xenograft model, suggesting that GSK3 ss promotes ESCC tumorigenesis primarily by driving SOX2 overexpression. GSK3 ss was found to be frequently overexpressed in clinical esophageal tumors, and there was a positive correlation between GSK3 ss and SOX2 protein levels. Notably, we found that SOX2 enhanced GSK3 ss expression transcriptionally, suggesting the existence of a vicious cycle that drives a coordinated GSK3 ss and SOX2 overexpression in ESCC cells. Finally, we demonstrated in tumor xenograft model that GSK3 ss inhibitor AR-A014418 was effective in suppressing SOX2-positive ESCC tumor progression and inhibited tumor progression cooperatively with chemotherapeutic agent carboplatin. In conclusion, we uncovered a novel role for GSK3 ss in driving SOX2 overexpression and tumorigenesis and provided evidence that targeting GSK3 ss may hold promise for the treatment of recalcitrant ESCCs.